Isotretinoin formulations and uses and methods thereof

ABSTRACT

Provided herein are novel isotretinoin formulations that provide an enhanced targeted dermal delivery system for the drug isotretinoin with improved thermodynamic activity using no to a small level of ethanol relative to existing isotretinoin gel products, and methods for treatment of ichthyosis and other skin conditions using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. national stage application is based on PCT InternationalApplication No. PCT/US2016/058746, filed Oct. 26, 2016, which claims thebenefit and priority in and to U.S. Provisional Application No.62/248,760, filed Oct. 30, 2015, and U.S. Provisional Application No.62/301,759, filed Mar. 1, 2016, which are both incorporated in theirentirety herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel isotretinoin formulations. Moreparticularly, the present invention provides an enhanced targeted dermaldelivery system for the drug isotretinoin with minimal to no systemicpenetration and improved thermodynamic activity using no ethanol or asmall level of ethanol relative to existing isotretinoin gel products,and methods for treatment of ichthyosis and other skin conditions usingthe same.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.This PCT International Application refers to various publications andpublicly available products, and the disclosures from such publicationsand products are incorporated in their entirety herein by reference.

Ichthyosis is a heterogeneous family of at least 28 mostly genetic skindisorders, characterized by dry, thickened, scaly or flaky skin,resulting from excessive aggregation of keratinocytes (abnormalcornification). Ichthyosis and related skin type disorders include abroad spectrum of conditions differing in onset (congenital to adultonset), etiology (inherited versus acquired), intensity (mild tosevere), and involvement (confined to the skin versus multisystem). Inmost, the barrier function of the skin is abnormal due to the abnormalprocess of epidermal maturation/differentiation, the quantity andquality of the stratum corneum, and keratinocyte proliferation kinetics.The American College of Osteopathic Dermatology estimates that 1 in 250individuals are affected by ichthyosis vulgaris, one of the more commontypes of ichthyosis. While several types can be acquired, most forms ofichthyosis are considered congenital. These types include, but notlimited to, Ichthyosis vulgaris, X-linked ichthyosis, Lamellarichthyosis, Congenital Ichthyosiform Erythroderma, Epidermolyticichthyosis, Erythrokeratodermia variablis, Pachyonychia congenital,Palmoplantar keratodermas, Harlequin type ichthyosis, Refsum disease,Conradi-Hunermann-Happle syndrome, CHILD syndrome, ichthyosis enconfettis, Epidermolytic nevus, Loricrin keratoderma, Voihwinkel'sdisease and Sjógren-Larsson syndrome. Current lifelong therapy consistsof emollients, including most standard ones which are poorly effectivefor treatment of severe ichthyosis, and keratolytic agents, such asalphahydroxy acids, produce burning sensations, especially in children.For congenital ichthyosis, however, there currently exist no FDAapproved therapies.

Isotretinoin is a retinoid, approved by the FDA for the treatment ofsevere recalcitrant nodular acne. Chemically, isotretinoin is13-cis-retinoic acid and is a derivative of vitamin A. Isotretinoin wasinitially developed and approved in 1982 for the treatment of acne.There are a number of ongoing studies regarding the use of isotretinoinfor treatment of musculoskeletal and connective tissue inflammations,emphysema, ulcerating diseases and various cancers, namely treatingcervical tumors in HIV positive women, the prevention of lung cancer insmokers and the prevention of skin cancer. Studies have been recentlycompleted or ongoing regarding the role of isotretinoin (usually incombination with other drugs) in the treatment of neuroblastoma,recurrent prostate cancer, leukemia, high-grade glioma, head and neckcancers and multiple myeloma. Isotretinoin has also been proved to beuseful in the treatment of certain dermatological conditions such asgram-negative folliculitis, recalcitrant rosacea, pyoderma faciale,generalized lichen planus, psoriasis, cutaneous lupus erythematosus,acne fulminans, and squamous cell carcinoma. It is also used for thetreatment of cutaneous photoaging. Further, isotretinoin has been usedin treating ichthyosis patients; however, this use has been limited tosystemic delivery via oral administration, which is burdened with acuteand chronic side effects to the patients, including birth defects,miscarriage, elevated cholesterol and triglycerides levels, skin drynessor stickiness, and pseudotumor cerebri. See, e.g., John J. DiGiovanna etal., “Systemic Retinoids in the Management of Ichthyoses and RelatedSkin Types,” Dermatol. Ther., Vol. 26(1): 26-38 (January-February 2013).

Isotretinoin was originally approved as a capsule, and it is still onlyavailable in this form in the United States. Isotretinoin formulationsas a gel or cream were also developed after the oral dosage form and areavailable in some countries outside the U.S. Additionally, a formulationknown as “Isotrexin,” comprising isotretinoin and erythromycin, has beenmarketed outside the United States. However, many topical formulationscomprise high concentrations of ethanol as a solvent and penetrationenhancer. High levels of ethanol, while aiding in drug permeability, cancause severe skin irritation and dryness, making many existingisotretinoin formulations unsuitable for use in treatment of skinconditions such as ichthyosis.

Therefore, there is a need for novel topical delivery systems ofisotretinoin that can safely be used for dermal administration withoutthe irritating effects of the existing topical formulations ofisotretinoin but maintaining the targeted administration of the drug.Further, there is a need for novel topical delivery systems ofisotretinoin in order to reduce or eradicate the risk of systemic sideeffects associated with isotretinoin administration.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery of novelformulations of isotretinoin that have no ethanol or significantly loweramounts of ethanol than presently marketed isotretinoin products butenhanced targeted local delivery of isotretinoin into the epidermis anddermis. Additionally, such novel formulations of isotretinoindemonstrate minimal to no systemic penetration.

In one aspect, the present invention comprises a pharmaceuticalcomposition comprising: isotretinoin; at least one penetration enhancer;at least one solvent; at least one viscosity modifying agent at leastone preservative; and water. In another aspect, an antioxidant can alsobe included. In another aspect, the pharmaceutical composition isselected from the group consisting of a gel, ointment, lotion, emulsion,cream, foam, mousse, liquid, spray, suspension, dispersion and aerosol.In another aspect, the at least one penetration enhancer can be selectedfrom the group consisting of ethanol, transcutol, and propylene glycol,or a combination thereof, and wherein the concentration of thepenetration enhancer is 0% w/w but no greater than 12.5% w/w. In anotheraspect, the antioxidant can be BHT, N-acetylcysteine (“NAC”), or acombination thereof. In a further aspect, the antioxidant can be presentin a concentration of 0.1%-30% w/w. In another aspect, the viscositymodifying agents can include cetyl alcohol, glycerol, polyethyleneglycol (“PEG”), or a combination thereof. In another aspect, the atleast one preservative can be selected from the group consisting ofmethyl parabens, propyl parabens, BHT, and a combination thereof. In yetfurther aspect, the solvent can be selected from the group consisting ofPEG, propylene glycol, Transcutol P, ethanol, and a combination thereof.The penetration enhancers can, in some aspects, be present in aconcentration of 0% w/w, 2% w/w, 5% w/w, or 10% w/w, or within the rangeof 0% w/w but no greater than 2% w/w. In a further aspect, theisotretinoin is present in a concentration selected from the groupconsisting of 0.025% w/w, 0.05% w/w, 0.10% w/w, 0.15% w/w, and 0.2% w/w.With respect to PEG, PEG can be any PEG of various molecular weightsknown in the art, such as those within the range of PEG 200 to PEG 4000.In a further aspect, the PEG can be a PEG (e.g., PEG 3350; PEG 4000), orcombination of PEGs (e.g., PEG 1450 & PEG 3350). In another aspect, atleast one humectant, which includes but not limited to urea or the like.In a further aspect, the at least one humectant can be present in aconcentration of 1%-40% w/w. In another aspect, an anti-inflammatorycompound can also be included. In a further aspect, theanti-inflammatory compound can be NAC, a steroid, a nonsteroidalanti-inflammatory compound, or a combination thereof. In a furtheraspect, the anti-inflammatory compound can be present in a concentrationof 0.003%-10% w/w.

In another aspect, a pharmaceutical composition is provided comprisingisotretinoin, PEG400, water, ethanol, methyl parabens, propyl parabens,PEG 4000, and BHT, wherein the concentration of ethanol is between 0%w/w and 10% w/w. The isotretinoin, in certain aspects, can be present ina concentration 0.025% w/w, 0.05% w/w, 0.10% w/w, 0.15% w/w, and 0.2%w/w; and in preferred aspects can be present in a concentration of 0.2%w/w.

In yet another aspect, a pharmaceutical composition is providedcomprising about 0.05% w/w to about 0.5% w/w isotretinoin, about 60% w/wto about 65% w/w PEG 400, about 6% w/w to about 12% w/w water, about 5%w/w to about 10% w/w ethanol, about 0.05% w/w to about 0.5% w/w methylparabens, about 0.01% w/w to about 0.05% propyl parabens, about 15% w/wto about 20% w/w PEG 4000, and about 0.05% w/w to about 0.2% w/w BHT.

In a still further aspect, the present invention provides apharmaceutical composition comprising about 0.2% w/w isotretinoin, about63.4% w/w PEG 400, about 9% w/w water, about 10% w/w ethanol, about 0.2%w/w methyl parabens, about 0.02% w/w propyl parabens, about 17.08% w/wPEG 4000, and about 0.1% w/w BHT.

In yet another aspect, a pharmaceutical composition is providedcomprising about 0.2% w/w isotretinoin, about 62.4% w/w PEG 400, about10% w/w water, about 5% w/w ethanol, about 5% w/w transcutol, about 0.2%w/w methyl parabens, about 0.02% w/w propyl parabens, about 17.08% w/wPEG 4000, and about 0.1% w/w BHT.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising about 0.1% w/w isotretinoin, about 62.4% w/w PEG400, about 10% w/w water, about 5% w/w ethanol, about 5% w/w transcutol,about 0.2% w/w methyl parabens, about 0.02% w/w propyl parabens, about17.08% w/w PEG 4000, and about 0.1% w/w BHT.

In another aspect, the present invention provides a method of treatingcongenital ichthyosis, comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising: isotretinoin; at least one penetration enhancer;at least one solvent; at least one preservative; and water; wherein theconcentration of ethanol is between 0% w/w and 10% w/w. In some aspects,the pharmaceutical composition can also comprise at least one componentselected from the group consisting of: an antioxidant, an emollient, aviscosity modifying agent, and a combination thereof. In some aspects,the administration can be dermal. In other aspects, the congenitalichthyosis is selected from the group consisting of Ichthyosis vulgaris,X-linked ichthyosis, Lamellar ichthyosis, Congenital IchthyosiformErythroderma, Epidermolytic ichthyosis, Erythrokeratodermia variablis,Pachyonychia congenital, Palmoplantar keratodermas, Harlequin typeichthyosis, Refsum disease, Conradi-Hunermann-Happle syndrome, CHILDsyndrome, ichthyosis en confettis, Epidermolytic nevus, Loricrinkeratoderma, Voihwinkel's disease and Sjógren-Larsson syndrome. Incertain aspects, the pharmaceutical composition comprises about 0.01%w/w to about 0.5% w/w isotretinoin, about 60% w/w to about 65% w/w PEG400, about 6% w/w to about 12% w/w water, about 5% w/w to about 12.5%w/w ethanol, about 0.05% w/w to about 0.5% w/w methyl parabens, about0.01% w/w to about 0.05% propyl parabens, about 15% w/w to about 20% w/wPEG 4000, and about 0.05% w/w to about 0.2% w/w BHT. In other aspects,the pharmaceutical composition comprises about 0.2% w/w isotretinoin,about 63.4% w/w PEG 400, about 9% w/w water, about 10% w/w ethanol,about 0.2% w/w methyl parabens, about 0.02% w/w propyl parabens, about17.08% w/w PEG 4000, and about 0.1% w/w BHT. In still other aspects, thepharmaceutical composition comprises about 0.2% w/w isotretinoin, about62.4% w/w PEG 400, about 10% w/w water, about 5% w/w ethanol, about 5%w/w transcutol, about 0.2% w/w methyl parabens, about 0.02% w/w propylparabens, about 17.08% w/w PEG 4000, and about 0.1% w/w BHT. In stillfurther aspects, the pharmaceutical composition comprises about 0.1% w/wisotretinoin, about 62.4% w/w PEG 400, about 10% w/w water, about 5% w/wethanol, about 5% w/w transcutol, about 0.2% w/w methyl parabens, about0.02% w/w propyl parabens, about 17.08% w/w PEG 4000, and about 0.1% w/wBHT. In a further aspect, the subject is human.

In another aspect, a pharmaceutical composition comprises isotretinoin,and at least one solvent. In a further aspect, the pharmaceuticalcomposition further comprises at least one penetration enhancer. In yetfurther aspect, the at least one penetration enhancer is selected fromthe group consisting of ethanol, transcutol, propylene glycol, and acombination thereof. In a further aspect, the at least one penetrationenhancer is ethanol, and the concentration of ethanol is no greater than2% w/w. In a further aspect, the pharmaceutical composition furthercomprises 0% w/w ethanol and/or 0% w/w water. In a further aspect, thepharmaceutical composition further comprises at least one preservative.In yet a further aspect, the at least one preservative is selected fromthe group consisting of methyl parabens, propyl parabens, BHT, and acombination thereof. In yet a further aspect, the at least one solventis selected from the group consisting of polyethylene glycol (PEG),propylene glycol, and a combination thereof. In a further aspect, thepharmaceutical composition is formulated as a gel, ointment, lotion,emulsion, cream, foam, mousse, liquid, spray, suspension, dispersion oraerosol.

In a further aspect, the pharmaceutical composition further comprises atleast one antioxidant. The at least one antioxidant can be NAC, BHT, ora combination thereof. In a further aspect, the concentration of NAC isat least 0.1% w/w but no greater than 30% w/w. In a further aspect, ananti-inflammatory compound can also be included, wherein theanti-inflammatory compound can be NAC, a steroid, a nonsteroidalanti-inflammatory compound, or a combination thereof. In yet a furtheraspect, the anti-inflammatory compound can be present in a concentrationof 0.003%-10% w/w.

In a further aspect, the pharmaceutical composition further comprises atleast one humectant. The at least one humectant can be urea, and theconcentration of urea can be at least 1% w/w but no greater than 40%w/w. In a further aspect, the pharmaceutical composition furthercomprises at least one viscosity modifying agent. The at least oneviscosity modifying agent can be glycerol, PEG, or a combinationthereof.

In another aspect, a pharmaceutical composition comprising isotretinoin,PEG 400, PEG 3350, and BHT. In a further aspect, the pharmaceuticalcomposition further comprises methyl parabens and/or propyl parabens. Ina further aspect, the isotretinoin is present in the concentration ofabout 0.01% to about 0.2% w/w. In a further aspect, the isotretinoin ispresent in the concentration of about 0.025% to about 0.2% w/w. In afurther aspect, the pharmaceutical composition further comprisesethanol, wherein the concentration of ethanol is no greater than 2% w/w.Alternatively, in a further aspect, the pharmaceutical compositioncontains 0% w/w ethanol. In a further aspect, the pharmaceuticalcomposition further comprises NAC.

In another aspect, a pharmaceutical composition comprising isotretinoin,PEG 400, PEG 3350, PEG 1450, and BHT. In a further aspect, thepharmaceutical composition further comprises methyl parabens and/orpropyl parabens. In a further aspect, the isotretinoin is present in theconcentration of about 0.01% to about 0.2% w/w. In a further aspect, theisotretinoin is present in the concentration of about 0.025% to about0.2% w/w. In a further aspect, the pharmaceutical composition furthercomprises ethanol, wherein the concentration of ethanol is no greaterthan 2% w/w. Alternatively, in a further aspect, the pharmaceuticalcomposition contains 0% w/w ethanol. In a further aspect, thepharmaceutical composition further comprises NAC.

In another aspect, a pharmaceutical composition comprising about 0.01%w/w to about 0.6% w/w isotretinoin, about 67% w/w to about 70% w/w PEG400, 0% w/w to about 2% w/w ethanol, about 0.2% w/w methyl parabens;about 0.02% w/w propyl parabens, about 14% w/w to about 30% w/w PEG3350, about 0% to about 15% w/w PEG 1450, and about 0.1% w/w BHT. In afurther aspect, wherein the amount of isotretinoin is about 0.025% toabout 0.6%.

In another aspect, a pharmaceutical composition comprising about 0.01%w/w to about 0.6% w/w isotretinoin, about 67% w/w to about 70% w/w PEG400, 0% w/w to about 2% w/w ethanol, about 14% w/w to about 30% w/w PEG3350, about 0% to about 15% w/w PEG 1450, and about 0.1% w/w BHT. In afurther aspect, wherein the amount of isotretinoin is about 0.025% toabout 0.6%.

In another aspect, a method of treating congenital ichthyosis,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprisingisotretinoin, and at least one solvent. In a further aspect, the atleast one solvent is 0% w/w but no greater than 2% w/w ethanol and/orPEG. In a further aspect, the pharmaceutical composition furthercomprises at least one antioxidant, wherein the at least one antioxidantis BHT, NAC, or a combination thereof. In a further aspect, theadministration is dermal. In a further aspect, the congenital ichthyosisis selected from the group consisting of Ichthyosis vulgaris, X-linkedichthyosis, Lamellar ichthyosis, Congenital Ichthyosiform Erythroderma,Epidermolytic ichthyosis, Erythrokeratodermia variablis, Pachyonychiacongenital, Palmoplantar keratodermas, Harlequin type ichthyosis, Refsumdisease, Conradi-Hunermann-Happle syndrome, CHILD syndrome, ichthyosisen confettis, Epidermolytic nevus, Loricrin keratoderma, Voihwinkel'sdisease, and Sjógren-Larsson syndrome. In a further aspect, the subjectis human.

In another aspect, a method of treating congenital ichthyosis,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition, the pharmaceuticalcomposition comprising about 0.01% w/w to about 0.6% w/w isotretinoin,about 67% w/w to about 70% w/w PEG 400, about 0% w/w to about 2% w/wethanol, about 0.2% w/w methyl parabens; about 0.02% w/w propylparabens, about 14% w/w to about 30% w/w PEG 3350, about 0% to about 15%w/w PEG 1450, and about 0.1% w/w BHT. In a further aspect, wherein theamount of isotretinoin is about 0.025% to about 0.6%. In a furtheraspect, wherein the amount of isotretinoin is about 0.025% to about0.2%. In a further aspect, the subject is human.

As used herein, “transdermal” administration means transport of an agentthrough or by way of the skin for introduction into systemiccirculation.

As used herein, “dermal” administration means transport of an agentacross the stratum corneum and into the dermis and/or epidermis fortreatment of a topical skin disorder (such as congenital ichthyosis)that responds to local, non-systemic administration of an agent. It willbe appreciated that some of the agent intended for dermal therapy can betransdermally administered, however typically not in an amountsufficient for therapy.

As used herein, “therapeutically effective amount” means an amount ofisotretinoin sufficient to prevent or reduce the symptoms associatedwith a disease or condition (such as congenital ichthyosis) and/orlessen the severity of the disease or condition. A therapeuticallyeffective amount is understood to be in context to the condition beingtreated, where the actual effective amount is readily discerned by thoseof skill in the art.

As used herein, “isotretinoin” refers to isotretinoin in the form of afree acid or its pharmaceutically acceptable salts, such as alkali metalsalts. Isotretinoin is 13-cis-retinoic acid. Tretinoin (all-transretinoic acid) and isotretinoin are geometric isomers and showreversible interconversion in vivo. The administration of one isomer cangive rise to another. Other major metabolites of isotretinoin such as4-oxo-isotretinoin and its geometrical isomer 4-oxo-tretinoin are alsocontemplated in the term “isotretinoin.”

As used herein, “permeation rate” means the rate of passage of the drugthrough the skin. Permeation rate is calculated as the slope of thelinear portion of the cumulative amount of drug permeated per cm² overtime.

As used herein, “transport rate” refers to the rate of passive drugtransport across human skin as governed by Fick's Law of diffusion. Themass transport equation is given as: J=1/A(dM/dt)=PΔC dt where J is flux(μg cm²/hr), A is cross sectional area of the skin membrane (cm²), P isthe apparent permeability coefficient (cm hr), ΔC is the concentrationgradient across the membrane, and (dM/dt) is the mass transport rate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic showing a Franz cell as used in the in vitro drugtransport experiments described herein, including a donor component, areceptor component, and a sampling side arm.

FIG. 2 is a line graph showing the mean cumulative amount ofisotretinoin (μg/cm2) delivered from a claimed composition (“PATPO3 AN”)as compared to Isotrex gel 0.05% (batches: F1311 and F0892), through asilicone membrane between 2 and 8 h (time represented as √time, mean±SE,5≤n≤6).

FIG. 3 is a line graph showing the percentage of applied dose ofisotretinoin delivered from PATPO3 AN and comparator product, Isotrexgel 0.05% (batches: F1311 and F0892), through silicone membrane between2 and 8 h (time represented as √time, mean±SE, 5≤n≤6).

FIG. 4 is a bar graph showing the percentage of applied dose ofisotretinoin recovered from skin strata (Stratum corneum, epidermis,dermis) and receiver fluid following the application of PATPO3 AN andcomparator product, Isotrex gel 0.05% (batch F0892), after the finalsampling point (48 h) (mean±SE, n=12).

FIG. 5 is a line graph showing the mean cumulative amount ofisotretinoin delivered per unit area (m/cm²) over a 24 h experimentalperiod through silicone membrane from PEG ointments and gel includingisotretinoin at 0.10%, compared to that from two batches of Isotrex gel0.05% (mean±SE, n≥5).

FIG. 6 is a line graph showing the mean cumulative amount per unit areaof isotretinoin (μg/cm²) delivered from PEG ointments and gel includingisotretinoin at 0.10% through silicone membrane between 1 and 8 h (timerepresented as √time), compared to that from two batches of Isotrex gel0.05% (mean±SE, n≥5).

FIG. 7 is a line graph showing the mean cumulative amount ofisotretinoin delivered per unit area (μg/cm²) over a 24 h experimentalperiod through silicone membrane from PEG ointments and gel includingisotretinoin at 0.20%, compared to that from two batches of Isotrex gel0.05% (mean±SE, n≥5).

FIG. 8 is a line graph showing the mean cumulative amount per unit areaof isotretinoin (m/cm²) delivered from PEG ointments and gel includingisotretinoin at 0.20% through silicone membrane between 1 and 8 h (timerepresented as √time), compared to that from two batches of Isotrex gel0.05% (mean±SE, n≥5).

FIG. 9 is a bar graph showing the mean cumulative amount of isotretinoinpermeated across the skin (μg/cm²) into the receiver fluid following theapplication of certain tested formulations and comparator product,Isotrex gel 0.05% (mean±SE, 10≤n≤12).

FIG. 10 is a bar graph highlighting the recovery of isotretinoin (μg)from the skin strata (Stratum corneum, epidermis, dermis) and receiverfluid following the application of certain tested formulations andcomparator product, Isotrex gel 0.05%, after the final sampling point(48 h) (mean±SE, 10≤n≤12). SC denotes Stratum corneum.

FIGS. 11A & 12B are chromatograms showing stability of the isotretinoinformulation PATPO3 AN containing 0.2% w/w isotretinoin, overlaid withPATPO3 AN placebo at t=0 weeks following storage at 40° C., between 2and 17 minutes.

FIGS. 12A & 12B are chromatograms showing stability of the isotretinoinformulation PATPO3 AN containing 0.2% w/w isotretinoin, overlaid withPATPO3 AN placebo at t=4 weeks following storage at 40° C., between 2and 17 minutes.

FIG. 13 is a bar graph showing recovery of isotretinoin (μg) from thesurface (residual formulation), skin strata (Stratum corneum, epidermis,dermis) and receiver fluid following the application of certain testedformulations and comparator product, Isotrex gel 0.05%, after the finalsampling point (48 h) (mean±SE, 10≤n≤12). SC denotes Stratum corneum.

FIG. 14 is a bar graph showing the cumulative amount of isotretinoinpermeated across the skin (μg/cm²) into the receiver fluid following theapplication of certain tested formulations and comparator product,Isotrex gel 0.05%, after final sampling point (24 h) (mean±SE, 10≤n≤12).

FIG. 15 is a bar graph showing the amount of isotretinoin penetratedthrough the skin (μg) from the surface (residual formulation), skinstrata (Stratum corneum, epidermis, dermis) and receiver fluid followingthe application of certain tested formulations and comparator product,Isotrex gel 0.05%, after the final sampling point (48 h) (mean±SE,10≤n≤12). SC denotes Stratum corneum.

FIG. 16 is a bar graph showing, with the surface removed, the amount ofisotretinoin penetrated through the skin (μg) from skin strata (Stratumcorneum, epidermis, dermis) and receiver fluid following the applicationof certain tested formulations and comparator product, Isotrex gel0.05%, after the final sampling point (48 h) (mean±SE, 10≤n≤12). SCdenotes Stratum corneum.

FIG. 17 is a bar graph showing, the amount of isotretinoin penetratedthrough the skin (μg) from skin strata (Stratum corneum, epidermis,dermis) and receiver fluid following the application of all PATPO3 AN(0.2%) (first and second data sets from the left) and Isotrex gel 0.05%(third and fourth data sets from the left), respectively, after thefinal sampling point (48 h) (mean±SE, 10≤n≤12). SC denotes Stratumcorneum. The first (PATPO3 AN) and third (Isotrex (0.05%)) data setsfrom the left are based on a fourth skin donor, while the second (PATPO3AN) and fourth (Isotrex F0892 (0.05%)) data sets are based on firstthree skin donors.

FIG. 18 is a table listing PEG ointment formulations for each of thepossible penetration enhancer options selected for the initialpermeation studies discussed further below. The penetration enhancercompositions, expressed as % w/w, of the PEG ointment formulations, areshown with the circles indicating those selected for the in vitropermeation and penetration experimentation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel targeted formulations ofisotretinoin that result in increased penetration of the dermal layerswith minimal to no systemic penetration and without increasing theconcentration of ethanol (or eliminating ethanol altogether) relative tothe presently marketed products, and methods of treatment of ichthyosisusing the same.

Dermal delivery of drugs provides many advantages; primarily, such ameans of delivery is a comfortable, convenient and noninvasive way ofadministering drugs. The variable rates of absorption and metabolismencountered in oral treatment are avoided, and other inherentinconveniences, e.g., gastrointestinal irritation and the like, areeliminated as well. Further, significant side effects associated withdelivery to other areas (e.g., to subdermal or extradermal structuresand/or to tissues other than dermis) are avoided.

Skin, however, is a structurally complex membrane. Molecules moving fromthe environment into and through intact skin must first penetrate thestratum corneum and any material on its surface. They must thenpenetrate the viable epidermis, the papillary dermis, and the capillarywalls into the blood stream or lymph channels. To be so absorbed,molecules must overcome a different resistance to penetration in eachtype of tissue. Transport across the skin membrane is thus a complexphenomenon. However, it is the cells of the stratum corneum, whichpresent the primary barrier to absorption of topical compositions ortransdermally administered drugs. The stratum corneum is a thin layer ofdense, highly keratinized cells approximately 10-15 microns thick overmost of the body. With many drugs, the rate of permeation through theskin is extremely low without the use of some means to enhance thepermeability of the skin.

Numerous chemical agents have been studied as a means of increasing therate at which a drug penetrates through the skin. As will be appreciatedby those in the field, chemical enhancers are compounds that areadministered along with the drug (or in some cases the skin can bepretreated with a chemical enhancer) in order to increase thepermeability of the stratum corneum, and thereby provide for enhancedpenetration of the drug through the skin. Ideally, such chemicalpenetration enhancers or “permeation enhancers,” as the compounds arereferred to herein, are compounds that are innocuous and serve merely tofacilitate diffusion of the drug through the stratum corneum. Thepermeability of many therapeutic agents with diverse physicochemicalcharacteristics can be enhanced using these chemical enhancement means.However, there are skin irritation, dryness and sensitization problemsassociated with high levels of certain enhancers, such as ethanol.

The novel formulations herein are surprisingly shown herein to haveimproved thermodynamic activity vis-a-vis the presently availableisotretinoin topical gel formulations, while having none or decreasedlevels of irritating ethanol agents. Typically, the influence of ethanolon drug permeability is concentration dependent, with the percutaneousabsorption being optimized at a level of greater than 50% w/w ethanol.In fact, the existing Isotrex gel products have over 95% w/w ethanol.The aspects of isotretinoin ointment formulations, though, threerepresentative formulations of which are shown in Table 1 below, eachcomprise less than 10% w/w ethanol. Thus, these novel formulationsenhance the delivery of isotretinoin into the epidermis and dermis, withlittle chance of causing skin irritation and dryness.

TABLE 1 % w/w % w/w % w/w Formulation 1 Formulation Formulation 3Component: (PATPO3 AN) 2 (PATPO6) (PATPO3 AI) Isotretinoin 0.2 0.2 0.1PEG 400 63.4 62.4 60.4 Water 9 10 10 Ethanol 10 5 0 Glycerol 0 0 5Propylene Glycol 0 0 7 Methyl Parabens 0.2 0.2 0.2 Propyl Parabens 0.020.02 0.02 PEG 4000 17.08 17.08 17.18 BHT 0.1 0.1 0.1 Transcutol 0 5 0Total 100 100 100

The novel preparations described herein are, in some aspects, formulatedas a gel, ointment, lotion, emulsion, cream, foam, mousse, liquid,spray, suspension, dispersion or aerosol, or any other vehicle known tothose of skill in the art. In preferred aspects, the preparations areformulated as a gel, ointment, foam, or cream.

A lotion can contain finely powdered substances that are insoluble inthe dispersion medium through the use of suspending agents anddispersing agents. Alternatively, lotions can have dispersed phaseliquid substances that are immiscible with the vehicle and are usuallydispersed by means of emulsifying agents or other suitable stabilizers.In some aspects, the lotion is in the form of an emulsion having aviscosity of between 100 and 1000 centistokes. The fluidity of lotionspermits rapid and uniform application over a wide surface area. Lotionsare typically intended to dry on the skin leaving a thin coat of theirmedicinal components on the skin's surface.

Creams can contain emulsifying agents and/or other stabilizing agents.In one aspect, the formulation is in the form of a cream having aviscosity of greater than 1000 centistokes, typically in the range of20,000-50,000 centistokes.

The basic difference between a cream and a lotion is the viscosity,which is dependent on the amount/use of various oils and the percentageof water used to prepare the formulations. Creams are typically thickerthan lotions, can have various uses and often one uses more variedoils/butters, depending upon the desired effect upon the skin. In acream formulation, the water-base percentage is about 60-75% and theoil-base is about 20-30% of the total, with the other percentages beingthe emulsifier agent, preservatives and additives for a total of 100%.

Examples of suitable ointment bases include hydrocarbon bases (e.g.,petrolatum, white petrolatum, yellow ointment, and mineral oil);absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin,and cold cream); water-removable bases (e.g., hydrophilic ointment), andwater-soluble bases (e.g., PEG ointments). Pastes typically differ fromointments in that they contain a larger percentage of solids. Pastes aretypically more absorptive and less greasy that ointments prepared withthe same components.

Some emulsions can be gels or otherwise include a gel component. Somegels, however, are not emulsions because they do not contain ahomogenized blend of immiscible components. Suitable gelling agentsinclude, but are not limited to, modified celluloses, such ashydroxypropyl cellulose and hydroxyethyl cellulose; Carbopolhomopolymers and copolymers; and combinations thereof. Suitable solventsin the liquid vehicle include, but are not limited to, diglycolmonoethyl ether; alklene glycols, such as propylene glycol; dimethylisosorbide; alcohols, such as isopropyl alcohol and ethanol. Thesolvents are typically selected for their ability to dissolve the drug.Other additives, which improve the skin feel and/or emolliency of theformulation, can also be incorporated. Examples of such additivesinclude, but are not limited, isopropyl myristate, ethyl acetate,C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone,capric/caprylic triglycerides, and combinations thereof.

Foams can include an emulsion in combination with a gaseous propellant.The gaseous propellant can include primarily of hydro fluoroalkanes(HFAs). Suitable propellants include HFAs such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixturesof these and other HFAs that are currently approved or can becomeapproved for medical use are suitable. The propellants preferably arenot hydrocarbon propellant gases which can produce flammable orexplosive vapors during spraying. Furthermore, the compositionspreferably contain no volatile alcohols, which can produce flammable orexplosive vapors during use.

The novel preparations are, in particularly preferred aspects,formulated as ointments. Ointments are semisolid preparations that aretypically based on petrolatum or other petroleum derivatives. Thespecific ointment foundation to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery,and, preferably, will provide for other desired characteristics as well,e.g., emolliency or the like. As with other carriers or vehicles, anointment foundation should be inert, stable, nonirritating andnonsensitizing. As explained in Remington: The Science and Practice ofPharmacy, 20^(th) edition (Lippincott Williams & Wilkins, 2000),ointment foundations can be grouped in four classes: oleaginous,emulsifiable, emulsion, and water-soluble. Oleaginous ointmentfoundations include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petroleum.Emulsifiable ointment foundations, also known as absorbent ointmentfoundations, contain little or no water and include, for example,hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.Emulsion ointment foundations are either water-in-oil (W/O) emulsions oroil-in-water (O/W) emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin and stearic acid. Preferred water-solubleointment foundations are prepared from PEGs of varying molecular weight.

Various additives, known to those skilled in the art, can, in someaspects, be included in the ointments. For example, solvents, includingrelatively small amounts of alcohol, can be used to solubilize certaindrug substances. Other optional additives include opacifiers,antioxidants, fragrance, colorant, gelling agents, thickening agents,stabilizers, surfactants and the like. Other agents can also be added,such as antimicrobial agents, to prevent spoilage upon storage, i.e., toinhibit growth of microbes such as yeasts and molds. Suitableantimicrobial agents are typically selected from the group consisting ofthe methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl andpropyl paraben), sodium benzoate, sorbic acid, imidurea, andcombinations thereof.

In some aspects, the ointments can also include penetration enhancingagents. Examples of classes of enhancers include, but are not limitedto, fatty acids, both saturated and unsaturated; fatty alcohols; bileacids; nonionic surfactants, including esters of fatty acids, fatty(long-chain alkyl or alkenyl) esters of monohydric alcohols, diols, andpolyols, diols and polyols that are both esterified with a fatty acidand substituted with a polyoxyalkylene, polyoxyalkylene fatty acidesters, polyoxyalkylene fatty ethers, polyoxyalkylene fatty ethers, andpolyglyceryl fatty acid esters; amines; amides;N-alkyl-azacycloalkanones and N-alkyl-azacycloalkenones; hydrocarbonsolvents; terpenes; lower alkyl esters; cyclodextrin enhancers;nitrogen-containing heterocycles; sulfoxides; and urea and itsderivatives.

Specific examples of suitable enhancing agents include ethers such asdiethylene glycol monoethyl ether (available commercially asTranscutol®, Gattefosse SA) and diethylene glycol monomethyl ether;surfactants such as sodium laurate, sodium lauryl sulfate,cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231,182, 184), Tween (20, 40, 60, 80) and lecithin; alcohols such asethanol, propanol, octanol, benzyl alcohol, and the like; fatty acidssuch as lauric acid, oleic acid and valeric acid; fatty acid esters suchas isopropyl myristate, isopropyl palmitate, methylpropionate, and ethyloleate; polyols and esters thereof such as PEG, and polyethylene glycolmonolaurate; amides and other nitrogenous compounds such as urea,dimethylacetamide, dimethylformamide, 2-pyrrolidone,1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine; terpenes; alkanones; and organic acids, particularlycitric acid and succinic acid. Azone® and sulfoxides such asdimethylsulfoxide and decylmethylsulfoxide can also be used, but areless preferred. Percutaneous Penetration Enhancers, eds. Smith et al.(CRC Press, 1995) provides an excellent overview of the field andfurther information concerning possible secondary enhancers for use inconjunction with the present invention.

EXAMPLES

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the exemplaryformulations and methods discussed herein.

Example 1 In Vitro Drug Transport Assessment

To assess the transport profiles of isotretinoin from certainformulations that included those described in Table 1, an in vitro drugtransport investigation was performed across a synthetic membrane. Thetransport of the drug from the selected formulations was compared usingmethod based on the principles of the FDA's SUPAC-SS guidelines [FDA(CDER), 1997, Guidance for industry—SUPAC-SS Non-sterile SemisolidDosage Form, Scale-up and post approval changes: Chemistry,manufacturing and controls; in vitro drug transport testing and in vivobioequivalence documentation].

A total of six formulations at each of the five concentrations ofisotretinoin (0.025, 0.05, 0.10, 0.15 and 0.20% w/w, were assessed (eachat n=6) herein and compared against the results of the comparatorproduct, Isotrex gel 0.05%. A total of two batches of Isotrex gel wereused in this experiment, which were both tested in six replicates.Furthermore, one batch of Isotrex gel 0.05% was tested concurrently in asubset of cells (n=3) as a control to assess the run-to-run methodvariability. The following parameters were employed: Receiver fluid: 2%Bruj in 20% ethanol: 80% PBS with 0.01% BHA; synthetic membrane:silicone; time points: t=0, 1, 2, 3, 4, 6, 7, 8, and 24 h; dose: greaterthan 0.3 g.

FIGS. 5 and 7 depict the transport of the drug from the claimedformulations at various concentrations of isotretinoin, compared to thatfrom Isotrex gel 0.05% (both batches, F0892 and F1311). The steady statetransport of isotretinoin across silicone membrane was seen from 1 hafter dosing, for all the formulations assessed. FIGS. 6 and 8 highlightthe steady state drug transport between 1 and 8 h (presented as a √time,as recommended in the SUPAC guidelines) from the tested prototypeformulations at various concentrations of isotretinoin, compared to thatfrom Isotrex gel 0.05%. The calculated transport rates (μg/cm2/√h) ofisotretinoin from all the formulations across silicone are reported inTables 2 and 3.

TABLE 2 Isotretinoin Transport Transport Transport Transport Transport %(w/w) 0.025% rate 0.05% rate 0.10% rate 0.15% rate 0.20% rateFormulations SSA37 7.74 ± SSA36 22.40 ± SSA33 40.52 ± SSA31 53.22 ±SSA24 48.62 ± 0.66  2.31 1.37 4.48 5.92 PATPO3 6.40 ± PATPO3  8.04 ±PATPO3 13.67 ± PATPO3 18.48 ± PATPO3 20.06 ± AF 1.38 n  0.43 AH 1.83 li3.29 AN 3.61 PATPO3 6.11 ± PATPO3  7.38 ± PATPO3 13.48 ± PATPO3 16.44 ±PATPO3 19.55 ± AA 1.02 AC  0.31 h 2.60 Ji 1.46 AO 2.00 PATPO3 4.46 ±PATPO3  6.72 ± PATPO3 12.80 ± PATPO3 13.58 ± PATPO6 18.55 ± AD 0.70 e 0.44 Ji 1.88 oi 1.99 3.42 PATPO3 3.47 ± PATPO3  5.43 ± PATPO3 11.36 ±PATPO5 12.91 ± PATPO3 17.65 ± AB 0.65 f  0.79 Al 1.51 1.15 gi 1.52PATPO3 3.28 ± PATPO3  5.00 ± PATPO3  9.91 ± PATPO3 12.80 ± PATPO3 13.77± fi 0.33 ii  0.88 ni 1.88 AG 1.88 hii 2.39

TABLE 3 Isotrex gel 0.05% Batch no. F1131 Batch no. F0892 19.09 ± 3.8617.30 ± 5.59

The statistical analysis of these results indicates that all theprototype gel formulations provided surprisingly and significantly(p≤0.05) greater transport rate of isotretinoin compared to Isotrex gel0.05%, with the exception of SSA37 (0.025%). For the PEG ointmentsincluding 0.1, 0.15 and 0.2% of isotretinoin, the transport of the drugwas observed to be similar (p>0.05) to that determined when usingIsotrex gel 0.05%. In contrast, the Isotrex gel was found to provide agreater transport with respect to the PEG ointments containing 0.025 and0.05% of isotretinoin, which is unsurprising considering that theIsotrex gel includes mainly ethanol, the evaporation of which causes thedrug thermodynamic activity, and so the flux, to increase over theduration of the experiment.

Example 2 In Vitro Permeation and Penetration Assessments

Following completion of the transport experiment, quantification ofisotretinoin on the surface of the skin and the skin strata wasperformed as described below:

Commonly, in vitro skin permeation experiments involve the use of adiffusion cell designed to mimic the physiological and anatomicalconditions of skin in situ. The model used in this experiment was theFranz diffusion cell as described in FIG. 1 (where the syntheticmembrane was replaced with human dermatomed skin). The subcutaneous fatwas removed mechanically and the skin was dermatomed to a thickness of400±100 μm using a Nouvag TCM 3000 cutter. Human dermatomed skin waspositioned between the donor and receptor compartment of a Franz cell(FIG. 1) with the Stratum corneum side up. For each active formulationup to 4 repetitions per formulation per skin donor (3 donors) wereperformed; however, only a single repetition was performed for theplacebo formulation (n=1).

FIG. 18 (which was Table 4 in the specification as filed) lists the PEGointment formulations for each of the possible penetration enhanceroptions selected for the initial permeation studies. The penetrationenhancer compositions, expressed as % w/w, of the PEG ointments, areshown with the circles indicating those selected for the in vitropermeation and penetration experimentation.

The following parameters were employed for the in vitro skin permeationexperiment:

Receiver fluid: 2% Brij in 20% ethanol: 80% PBS with 0.01% BHA.

Time points: t=0, 6, 24 and 48 h.

Membrane: human dermatomed skin with a thickness of 400±100 μm (fromthree different donors).

Dose: ˜10 mg/cm²).

The following procedures were employed: Franz diffusion cells with anaverage surface area of approximately 0.6 cm² and a volume ofapproximately 2.0 mL were employed. First, prior to dosing, theintegrity of the skin was assessed as follows: (a) Dermatomed skin (fromthree donors) was mounted between the donor and receiver compartmentsand the cells were sealed together using Parafilm® and clips. (b) Thedonor and receiver chambers were filled with PBS solution and a smallmagnetic follower was placed in the receiver compartment. (c) Cells wereequilibrated in a water bath ensuring a membrane temperature of 32° C.for 30 min (water bath temperature of 37° C.). (d) The resistance of theskin in each Franz cell was measured using the LCR 6401 Databridge (SOP3118). (e) The electrodes were placed in the receiver compartmentthrough the sampling arm and the donor chamber. (f) The LCR was set at100 Hz and set to ‘R’ for resistance. (g) Cells with a resistance belowthe acceptable limits were discarded and remounted. Acceptable limitsare defined according to the measurement of controls for dermatomedskin, where the skin has been deliberately perturbed. Cells with greaterthan twice the resistance (KΩ) of the control were considered acceptableand selected for the Franz cell permeation experiment.

Second, following skin integrity testing the PBS solution was removedfrom each compartment and the receiver compartment of acceptable cellswas filled with receiver fluid (2% Brij in 20% ethanol: 80% PBS with0.01% BHA). Each cell was then equilibrated to ensure a surfacetemperature of 32° C. (external skin surface temperature) for at least30 min prior to dosing (water bath temperature of 37° C.). Third, apositive displacement pipette was used to apply the formulation (˜8 mg)to the plunger of a 1 mL syringe. The formulation (6-7 mg) was appliedto the skin surface and spread over the diffusion area using theplunger. Prior to and after application the weight of the plunger wasrecorded and the amount per cell was calculated. Fourth, receiver fluid(200 μL) was removed at the following time points t=0, 6, 24 and 48 hand transferred to a HPLC vial for analysis using HPLC. Fifth, freshpre-warmed receiver fluid (200 μL) was used to replace the receiverfluid removed at each time point. Sixth, following the final time point(48 h), the Franz cells were dismantled and the drug was recovered fromthe skin.

Isotretinoin was recovered from the surface of the skin as follows: (i)After dismantling the donor chamber from the Franz cell, one dry cottonswab was used to remove any residual formulation from the surface of theskin and the swab placed into the 7 mL vial; (ii) A second swab wasimmersed into the extraction diluent (90:10-ethanol:water) and used toswab the surface of the skin; this swab will then be placed into thevial containing the first swab; (iii) The final swab was used dry toswab the surface of the skin and then placed into the glass vialcontaining the two other swabs; (iv) An initial tape strip (usingD-Squame®) from the surface of the skin was also placed in with thecotton wool swabs and 2 mL of extraction diluent (90:10 ethanol:water)was added; (v) Each vial was then shaken on an orbital shaker at ambienttemperature for at least 16-20 h in the extraction solvents tofacilitate the extraction; (vi) Following the extraction procedure, theextraction diluent was removed from the vials and centrifuged at 13,000rpm for 10 min to remove all un-dissolved materials and particles; and(vii) The supernatant from each sample was then transferred to a HPLCvial and analysed using HPLC.

The following procedure was then used to recover isotretinoin from theStratum corneum: (i) A total of five further tape strips (usingD-Squame®) from the surface of the skin were taken, placed into a glassvial and 2 mL of extraction diluent (90:10-ethanol:water) was added toit; (ii) The vials from Step (i) were shaken on an orbital shaker atambient laboratory temperature for 16 to 20 h; (iii) Following theextraction procedure (Step (ii)), the extraction solution was removedfrom the vials and centrifuged at 13,000 rpm for 10 min to remove allun-dissolved materials and particulates; and (iv) The supernatant wasplaced into vials for analysis by HPLC.

As described in greater detail below, the remaining epidermis and dermiswere processed as follows: (i) The remaining epidermis (after removingthe Stratum corneum by using 5 tape strips) was heat-separated from thedermis by dry heating at 60 degrees Celsius for 2 minutes; (ii) Theepidermal and dermal layers were placed into individual glass vials and2 mL of extraction solvent (90:10-ethanol:water) was added to thesevials; (iii) The vials from Step (ii) were shaken on an orbital shakerat ambient laboratory temperature for 16 to 20 h; (iv) Following theextraction procedure (Step (iii)), the extraction solution was removedfrom the vials and centrifuged at 13,000 rpm for 10 min to remove allun-dissolved materials and particulates; and (v) The supernatant wasplaced into vials for analysis by HPLC.

The data was interpreted as follows: (i) Data was manually transcribedinto Excel spreadsheets. The levels of isotretinoin detected in thereceiver fluid and skin strata were calculated from the respectivecalibration standards; (ii) The total amount (μg) of isotretinoin pervolume sampled was calculated (total amount/volume sampled=μg/mL×volumesampled); (iii) The total amount (μg) of isotretinoin recovered at eachtime point as then calculated (total amount=μg/mL×total volume of eachFranz cell); (iv) The cumulative amount (μg) of isotretinoin wascalculated by adding the total amount (μg, Step (iii)) at each timepoint with the total amount withdrawn (μg) from each of the previoustime points (Step (ii)); (v) The cumulative amount per unit area ofisotretinoin (μg/cm2) was calculated by dividing the cumulative amount(μg, Step (iv)) by the diffusion area (μg/cm2=cumulative amount(μg)/diffusion area); (vi) Any outliers were rejected according tointernal procedures; (vii) The transport rate of isotretinoin acrosssilicone membrane from all the formulations tested was calculated asslope of the linear portion of the profile “cumulative amount permeatedof drug permeated per cm2 vs. √t (as recommend per SUPAC guidelines);and (viii) The total amount (μg) of isotretinoin in each of the skinmatrices was calculated (total amount=μg/mL×extraction dilution).

Statistical analysis of data was performed using statistical package forsocial science (SPSS) version 19.0 (SPSS Inc., USA). Such analysis wascarried out to determine any significant difference in the amount ofisotretinoin released from each test formulations and comparatorproduct. Furthermore, statistical analysis was carried out to identifysignificant difference in the levels of drug recovered from surface,Stratum corneum, epidermis and dermis and receiver fluid at 48 h, fromeach of the tested formulations compared to the Isotrex gel 0.05%.

A total of 15 formulations were selected in addition to the comparatorproduct (Isotrex gel 0.05%) for permeation and penetration analysis.FIG. 9 shows the amount of drug permeated across the skin into thereceiver fluid at 6, 24 and 48 h, following application of all theformulations to the surface of the skin at T=0 h. FIG. 13 depicts thepenetration profiles of the drug (using human skin from three donors)from all the prototype formulations investigated, at each of the fiveconcentrations of isotretinoin, compared to that from Isotrex gel 0.05%(batch F0892). FIG. 10 highlights the amount of isotretinoin penetratedinto the skin layers and receiver fluid, with the exclusion of theamount of drug recovered from the surface of the skin (residualformulation). The analysis of the permeation data showed that the amountof drug recovered in the receiver fluid was below the LOQ for most ofthe formulations tested, at 6 and 24 h. Exclusions to this trend wereobserved when the drug was formulated as PATPO3 AF 0.025%, PATPO3 h0.10%, PATPO5 0.15%, PATPO3 AG 0.15%, PATPO3 AO 0.20%, SSA36 0.05%,SSA31 0.15% and SSA24 0.20%, where isotretinoin was detected in thereceiver fluid (FIG. 9) at levels above the LOQ. The drug was recoveredin the receiver fluid at 48 h, following the application of all theformulations, with the exception of PATPO3 N 0.05%, PATPO3 AI 0.10%.

The analysis of the penetration data showed that as a general trend thehighest amount of isotretinoin was recovered from the surface of theskin after the application of all the tested formulations, with thehighest amount of isotretinoin recovered being 4.78±0.66 μg (SSA24,0.20%). The highest delivery of isotretinoin into the epidermal layer(site of action of the drug) was observed with the application of PATPO50.15% (0.29±0.10 μg), PATPO6 0.20% (0.29±0.09 μg) and PATPO3 AN 0.20%(0.28±0.10 μg), which provided levels of isotretinoin approximately10-fold and significantly (p≤0.05) higher compared to those observedwith Isotrex gel, 0.05% (0.03±0.01 μg).

The analysis of the data related to the amount of isotretinoin recoveredfrom the dermis indicate that the formulations providing the highestlevels of the drug into this skin layer were PATPO3 AN 0.20% (0.54±0.15μg) and PATPO3 AO 0.20% (0.47±0.12 μg), which again were found to be ca.10-fold and significantly higher (p≤0.05) compared to those achievedwith Isotrex gel, 0.05% (0.05±0.01 μg). The highest level ofisotretinoin recovered from the Stratum corneum was observed after theapplication of the comparator product Isotrex gel (0.64±0.25 μg),followed by SSA24 (0.58±0.09 μg) and PATPO6 0.20% (0.44±0.10 μg).

Interestingly, as noted in EXAMPLE 1, the difference in the transport ofisotretinoin across silicone from PATPO3 AN and Isotrex gel (FIG. 3) didnot manifest into a similar relationship for delivery to the skin asshown in FIG. 4, where a 2-3 fold increase in the percentage ofisotretinoin penetrated into the skin strata (epidermis and dermis) wasobserved following the application of PATPO3 AN (when expressed as % ofapplied dose). When the data was expressed as cumulative amount of drugpermeated into and across the skin (μg/cm²), the enhancement in thedelivery of isotretinoin from PATPO3 AN to epidermal and dermal layerswas measured to be approximately 10-fold, compared to Isotrex gel. Thedrug delivery profile of PATPO3 AN appears to be unique in that thedelivery of drug appears to be targeted to the pathological site(epidermis/dermis) (despite the low levels of ethanol) unlike theIsotrex gel which has a more typical profile where delivery is focusedto the Stratum corneum (FIG. 3). Thus, PATPO3 AN provides a uniquetargeted dermal delivery system that nevertheless resists systemicdelivery.

Example 3 Stability Assessments

Following completion of the permeation/penetration experiment,accelerated stability testing on PATPO3 AN formulations containing0.025% and 0.2% w/w isotretinoin, as set forth below in Table 5, whereX=an isotretinoin assay and related substances, visual appearance,microscopic observations, and apparent pH; and A=backup.

TABLE 5 Storage conditions Initial t = 0 2 weeks 4 weeks 2-8° C.  X A X25° C. X X 40° C. X X

Representative batches of PATPO3 AN containing 0.2% w/w isotretinoin insealed containers were stored at 2-8° C. (the stability of isotretinoinat this temperature was tested at 4 weeks only), 25° C. and 40° C. forfour weeks and then tested using HPLC with n=3 replicates at t=2 and t=4weeks. A PATPO3 AN placebo was tested for baseline comparison. Theresults are shown in FIGS. 11 and 12, which provide a representativechromatogram of PATPO3 AN containing 0.2% w/w isotretinoin, as comparedagainst a placebo, between 2 and 17 minutes, at t=0 and t=4 weeks,respectively.

In addition, based on physical stability experiments (e.g., storage at25° C. for up to 5 weeks after manufacture (with two freeze thawcycles—between 2-8 and 25° C.) prior to assessment; centrifuge testranging from 2-16 minutes of centrifuging), formulations with 0% to 2%w/w of ethanol show surprisingly improved physical stability (withreduced susceptibility to syneresis) during centrifuge tests relative toequivalent formulations with a higher ethanol content. Also,formulations with PEG 3350 shows improved physical stability (withreduced susceptibility to syneresis) relative to equivalent formulationsprepared with PEG 4000.

Glycerol appears to improve physical stability where syneresis informulations with propylene glycol occurs to a greater degree in 8%glycerol versus 15% glycerol formulations Here, only a very smalldroplet of liquid was observed, following centrifugation for 12-15minutes, and could be distinguished from the main formulation byplacement of glass pipette at the surface of the formulation.

Example 4 In Vivo Assessment

To evaluate the toxicity of the formulations herein, threeconcentrations of PATPO3 AN were administered daily as a topicalointment to miniature swine (Sus scrofa) for 90-days, followed by a28-day recovery period. In addition, the toxicokinetic (“TK”)characteristics of PATPO3 AN were evaluated.

Forty-eight (48) Hanford miniature swine were assigned to 5 groups, with4 animals per gender per group in the main cohort and two additionalanimals per gender per group in the vehicle and high dose groups for therecovery cohort. Groups 1 through 5 were the sham, vehicle, low (0.1mg/kg), mid (0.2 mg/kg) and high (0.4 mg/kg) dose groups, respectively.The dose of 0.2 g/kg was applied to a single site for each animal andwas adequate to uniformly cover the entire 10% body surface area with athin layer of PATPO3 AN. The size of the site (approximately 10% of thetotal body surface area for each animal) was based on the followingformula:10% Total body surface area (cm²)=9.533 [BW (grams)]^(2/3)×0.10

The average body surface area for each gender was used to determine theapproximate 10% body surface for each study animal. Dose sites wereclipped with electric clippers (blade no. 40 or finer) between Day −3and Day −1, and at least the corners of the dose sites were marked witha permanent marker. Dose sites were re-clipped and re-marked throughoutthe study period as necessary. The size of the dose sites were adjustedthroughout the study as needed. Prior to each dose administration, thedose site was washed with water soaked gauze and then dried with drygauze. Prior to dose administration each day, tubes containing testarticle and vehicle used on that day were shaken at least 5 times.Animals were dosed (by weight) with the appropriate vehicle or testarticle daily, which remained in place for 24 hours±2 hours.

Animals were topically treated once per day for 90 days, followed by a28-day recovery period. At designated time points, blood samples werecollected for toxicokinetic (“TK”) analysis. On Day 91, the main cohortanimals were necropsied with tissue collection, followed by recoverycohort animals on Day 118.

Animals were evaluated for signs of toxicity through physicalexaminations, clinical observations, body weight, body weight change,dose site Draize scoring, clinical pathology (hematology, coagulation,serum chemistry and urinalysis), electrocardiography, ophthalmology,gross pathology, organ weight and histopathology. Toxicokineticcharacteristics were assessed on study Day 1 and Day 90.

The study design, parameters evaluated, and TK sample collection schemeare presented in Tables 6, 7 and 8, respectively.

TABLE 6 Study Design Number of Animals Application IsotretinoinIsotretinoin Treatment Test Main Recovery Rate g/kg Conc. Dose LevelGroup Article Male Female Male Female (mL/kg ^(a)) mg/g (%) (mg/kg) 1N/A 4 4 N/A N/A N/A N/A N/A (Sham) 2 Vehicle 4 4 2 2 0.2 (0.19) N/A   0(Vehicle) 3 PATP03 4 4 N/A N/A 0.2 (0.19) 0.5 0.1 (Low) AN (0.05%) 4 4 4N/A N/A 0.2 (0.19) 1.0 0.2 (Mid)  (0.1%) 5 4 4 2 2 0.2 (0.19) 2.0 0.4(High)  (0.2%) Study Day 1 corresponds to the first day of doseadministration. ^(a) Formulation density = 1.068 g/mL

TABLE 7 Parameters Evaluated and Intervals Parameters ApproximateIntervals Mortality/ At least twice daily Moribundity observationsPhysical During acclimation examination Clinical Once during acclimationobservations Prior to each dose on Days 1-90 Then daily thereafter onDays 91-118 Draize score Once during acclimation (dose site) Prior toeach dose on Days 1-7 Then weekly thereafter Body weights Prior torandomization, prior to first dose, weekly thereafter and prior totermination Ophthalmology During acclimation (main & recovery), prior totermination of main cohort (main & recovery), and prior to terminationof recovery cohort Clinical Pathology During acclimation (main &recovery), prior to (hematology, termination of main cohort (main &recovery), serum chemistry, and prior to termination of recovery cohortcoagulation, & urinalysis ECG During acclimation (main & recovery),prior to termination of main cohort (main & recovery), and prior totermination of recovery cohort Toxicokinetics Day 1 and 90Necropsy/organ Day 91 main animals and Day 118 recovery animals weightsHistopathology Dose site and control skin on all animals (main andrecovery), standard tissues on all study animals (main & recovery) inall groups, including gross lesions (if any)

TABLE 8 TK Blood Collection Scheme Study Day Target Sample CollectionTime Points  1 (Groups 1-5) Pre, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24hours postdose 90 (Groups 1-5) Pre, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24hours postdose

Blood samples were collected into tubes containing K₂-EDTA as theanticoagulant. Plasma samples were prepared by centrifuging at ˜3000 rpmfor approximately 15 minutes at ˜4° C. All of the plasma samples werealiquoted in a single container and frozen on dry ice. The plasmasamples were stored in a freezer at approximately −70° C. until shipmenton dry ice to the analytical lab for analysis. All samples from allanimals were collected according to Table 8 on Days 1 and 90. Groups 1and 2 only had the 2-hour post-dose sample analyzed for isotretinoin andtretinoin for Days 1 and 90. Groups 3-5 had all samples analyzed forisotretinoin and tretinoin for Days 1 and 90.

Animals, housing and environmental conditions used for this assessmentis found in Table 9.

TABLE 9 Animals, Housing and Environmental Conditions Species: Susscrofa, miniature swine Strain: Hanford, naïve Source: SinclairBio-Resources, LLC Age at Acclimation: 2.5-4.0 months Weight at Week-1:9.5-17.3 kg Number and Gender: 48 (24 males and 24 females)Identification: Numbered ear tag and cage card Acclimation: At least 14days Caging: Animals were housed in ~3′ × 5′ stainless steel solid lowerwalls with upper vertical bars and front gate and elevated PVC-coatedexpanded metal flooring Enrichment A steel chain suspended by a rope wasprovided in each pen for animals as enrichment Number per cage: 1Environmental Temperature: 20.3° C. to 26.4° C. conditions: (68.5° F. to79.5° F.) Photoperiod: 12-hr light/12-hr dark

Detailed clinical observations were made once during acclimation, priorto dosing on Days 1-90, and then daily thereafter from Days 91-118. Thedose sites were observed and scored for skin irritation once duringacclimation, prior to each dose on Days 1-7, and then weekly thereafteruntil study termination. Local findings at the dose sites were scoredusing a modified Draize-scoring system (Table 10) to determine thedegree of inflammation (erythema and edema).

TABLE 10 Draize Scoring System Category Score Description Erythema 0 Noerythema 1 Slight erythema 2 Well-defined erythema 3 Moderate or severeerythema 4 Severe erythema or slight eschar formation (injuries indepth) Edema 0 No edema 1 Very slight edema 2 Slight edema (well-definededges) 3 Moderate edema (raised >1 mm) 4 Severe edema (raised >1 mm andextending beyond the area of exposure)

Ophthalmology examinations were performed during acclimation (main andrecovery cohorts), prior to main cohort termination, and prior totermination of recovery cohort. A board-certified veterinaryophthalmologist performed all ocular examinations. The examinationsincluded, but were not limited to, the conjunctiva, cornea, anteriorchamber, iris, lens, vitreous humor, retina, and ocular fundus. Anappropriate mydriatic agent was administered prior to the examination.

Electrocardiography (ECG) were performed on all study animals duringacclimation (main and recovery cohorts), prior to main cohorttermination, and prior to recovery cohort termination. The animals' hairat the sites to which the electrodes were attached was clipped andmoistened with gel before placement. The appropriate electrodes wereattached according to the electrocardiogram machine manufacturer'sinstructions with settings consistent with conventional veterinaryprocedures. Recordings of the standard leads I, II, and III werecollected at paper speeds of 25 mm/sec and 50 mm/sec with a minimum ofthree complexes for each recording. ECG recording times were for 20 to60 seconds at 25 mm/sec to assess arrhythmias and a brief tracing at 50mm/sec for ease of measurement of P-QRS-T waveforms The ECG data wassubmitted to a board-certified veterinary cardiologist forinterpretation of qualitative parameters (i.e., abnormalities in rhythm,conduction, etc.) and quantitative data, including, but not limited to:HR, RR, PR, QRS, QT and QTc intervals.

For clinical pathology, blood samples were collected from study animalsfor clinical pathology tests during acclimation (main and recoverycohorts), prior to main cohort termination (main and recovery cohorts),and prior to recovery cohort termination.

Blood samples (˜2 mL/animal) were collected into a tube containingK₃-EDTA as anticoagulant. Samples were stored on wet ice or refrigeratedat ˜4° C. until analyzed. The hematology analysis included:

White blood cell count Neutrophils (% and absolute) Red blood cell countEosinophils (% and absolute) Hemoglobin Basophils (% and absolute)Hematocrit Lymphocytes (% and absolute) Mean cell volume Monocytes (%and absolute) Mean cell hemoglobin Platelet count Mean cell hemoglobinconcentration Reticulocytes Differential white blood cell count

Blood samples (˜1.8 mL/animal) were collected into tubes containingsodium citrate (3.2%). Plasma was prepared by centrifuging for ˜15minutes at ˜3000 rpm at ˜4° C. Plasma was stored temporarily on wet iceor refrigerated at 4° C. until analyzed. The coagulation analysisincluded:

Activated partial thromboplastin time (APTT) Prothrombin time (PT)Fibrinogen

With respect to serum chemistry Blood samples (˜2 mL/animal) werecollected from each animal, into tubes without anticoagulant. Blood wasallowed to clot before being centrifuged for ˜15 minutes at ˜3000 rpm at˜4° C. and serum was harvested into a polypropylene cryovial. Serum wasstored temporarily on wet ice or refrigerated at 4° C. until analyzed.The serum chemistry analysis included:

Alanine aminotransferase (ALT) Creatinine Albumin GlobulinAlbumin/Globulin ratio Glucose Alkaline phosphatase (ALP) Inorganicphosphorus Aspartate aminotransferase (AST) Potassium Blood UreaNitrogen (BUN) Sodium Calcium Total bilirubin Chloride Total proteinCholesterol Triglycerides

Urine (˜2 mL if available) was collected using metabolism cagesovernight at room temperature. The sample was placed in a sterile redtop tube without anticoagulant and stored on wet ice or refrigerated at˜4° C. until analyzed. The samples were analyzed for the followingparameters:

Bilirubin Protein Blood Specific gravity Color and clarity UrobilinogenGlucose Urine microscopic examination including the Ketones presence ofWBC, RBC, crystals and bacteria pH

At scheduled sacrifices, the following organs (when present) wereweighed, with paired organs weighed together. Relative organ weights(organ-to-body weight and organ-to-brain weight) ratios were calculated:

adrenal (2) pituitary gland Brain spleen epididymis (2) testis (2) Heartthymus kidney (2) thyroid liver with gall bladder (drained) uterus

The following tissues (when present) from all animals were preserved in10% neutral-buffered formalin (except where noted):

adrenal (2) ovary (2) with oviduct aorta pancreas bone (femur & sternumwith marrow) pituitary gland bone marrow smear*** rectum brain(cerebellum, cerebrum, medulla & pons) salivary gland [mandibular (2)]cecum sciatic nerve cervix seminal vesicle (2) colon skeletal muscle(quadriceps femoris) duodenum skin—abdomen epididymis (2) skin—from dosesite with underlying tissue esophagus skin—from non-dose site withunderlying tissue eyes (2) with optic nerve* spinal cord (cervical,thoracic & lumbar) heart spleen ileum stomach jejunum testis (2)* kidney(2) thymus Lacrimal gland thyroid (2) lesions** tongue liver withgallbladder (drained) trachea lung with main stem bronchi urinarybladder lymph nodes (mandibular) uterus lymph node (mesenteric) vaginamammary gland (females)  *Eyes and testes were fixed in Davidson's andModified Davidson's, respectively for 1-3 days and then both were storedin 70% alcohol.  **Gross lesions were collected. ***Bone marrow smearswere fixed in methanol and retained for evaluation at the discretion ofthe Study Director in consultation with the study pathologist andSponsor.

All preserved tissues were submitted to Histo-Scientific ResearchLaboratories (“HSRL”). Main and Recovery cohort animals had preservedtissues embedded in paraffin, sectioned, stained with hematoxylin andeosin, and examined microscopically by a board-certified veterinarypathologist. Gross lesions were examined microscopically. Followinghistopathology evaluations, all prepared slides, remaining wet tissues,blocks, and raw data were returned for archive.

Once daily topical (dermal) application of PATP03 AN to 10% of the totalbody surface of miniature swine for 90 days at up to 0.4 mg/kg/dayresulted in no toxicologically meaningful effects onmortality/moribundity, non-dermal clinical observations, body weightgain, ophthalmologic or electrocardiographic examinations, hematology,serum chemistry or urinalysis parameters, or organ weights. There wereno unscheduled deaths or significant moribundity for any animal. Dermaleffects observed were consistent with the well-known characteristics oftopically applied retinoids.

Analysis of plasma isotretinoin/tretinoin parameters showed minimalisotretinoin systemic exposure following a single PATP03 AN applicationwith tretinoin exposure greater than that of isotretinoin. With repeatPATP03 AN dermal administration, isotretinoin exposure plateaued at themid-dose level (0.2 mg/kg). Isotretinoin accumulation was evident, butwithout an associated increase in tretinoin exposure. A systemic NoObservable Adverse Effect Level (“NOAEL”) of 0.4 mg/kg/day wasidentified (associated with a Day 90 isotretinoin AUC values of 44.3hr*ng·mL).

Example 5 Additional Formulations

Table 11 lists four formulations at 0.2% w/w of isotretinoin and twoformulations at 0.6% w/w of isotretinoin were assessed herein andcompared against results of the comparator product, Isotrex gel 0.05%.Unlike Isotrex gel that has over 95% ethanol, the six formulations hadno or much lower concentration (e.g., 2%) of ethanol, respectively.Additionally, five of the six formulations (EtOH 2%/PEG3350; PATPO3 PEGonly with 1450-b; PATPO3 f-d, 5% glycerol, PEG3350; EtOH 2%/PEG3350; andPATPO3 PEG only with 1450-b) did not include water.

TABLE 11 Target composition % w/w PATPO3 PATPO3 f-d, PATPO3 EtOH 2%/Water 5%/ PEG only 5% Glycerol, EtOH 2%/ PEG only PEG3350 PEG3350 with1450-b PEG3350 PEG3350 with 1450-b Excipient (0.2% API) (0.2% API) (0.2%API) (0.2% API) (0.6% API) (0.6% API) SR PEG 67.58 65.38 69.3 69.7 67.1869.3 400 Water — 5 — — — — Glycerol — — — 5 — — Ethanol 2 — — — 2 —Methyl 0.2 0.2 0.2 0.2 0.2 0.2 parabens Propyl 0.02 0.02 0.02 0.02 0.020.02 parabens PEG 4000 — — — — — — PEG 3350 29.9 29.1 14.78 14.78 29.914.78 PEG-1450 — — 15 10 — 15 BHT 0.1 0.1 0.1 0.1 0.1 0.1 Isotretinoin0.2 0.2 0.2 0.2 0.6 0.6 Total 100 100 100 100 100 100 (—): not included.

FIGS. 14 and 15 show results using the six formulations listed in Table11 against the comparator product, Isotrex gel 0.05% using the same invitro skin permeation and penetration experiment parameters, proceduresand data calculations discussed in EXAMPLE 2 above. However, thedermatoned skin used for these experiments came from a fourth donor. Asshown in FIG. 14, isotretinoin was not found in the receiver fluid forthe t=0 and 6-hour time points for any of the formulations tested.However, after 24 hours, isotretinoin is found in the receiver fluid inminimal amounts in the tested formulations, including Isotrex and PATPO3AN. These results show greater permeability when comparing the resultsin FIG. 9, which shows that isotretinoin in receiver fluid did not occuruntil 48 hours. This may be due to greater permeability from the fourthdonor than the three donors used during the previous assessmentdiscussed earlier. See also FIG. 17, which shows a comparison of twoskin penetration data sets of PATPO3 AN (0.2%) and Isotrex (0.05%),respectively, and that the first and third data sets from the left,which both are based on the fourth donor, show greater permeability thanthe second and fourth data sets from the left, which both are based onthe three previous donors.

FIG. 16 shows that the following formulations demonstrate unexpected,superior delivery of isotretinoin to the epidermis and dermis thanIsotrex gel: PATPO3 AN; EtOH 2%/PEG3350; PATPO3 PEG only with 1450-b;and PATPO3 f-d, 5% glycerol, PEG3350. Again, these formulations havemuch a lower concentration of to no ethanol, which is in severe contrastto the more than 95% ethanol in Isotrex gel. Formulations prepared at0.6% w/w do not appear to deliver more isotretinoin than formulationsprepared at 0.2% w/w. Hence, the novel formulations disclosed hereinshow unexpected, superior delivery efficiency of isotretinoin to theepidermis and dermis, and that such delivery efficiency is not simply aresult of increasing the amount of isotretinoin in the formulation.Indeed, FIG. 15 confirms that a large amount of isotretinoin remained atthe skin surface for the 0.6% w/w isotretinoin formulations (see, e.g.,FIG. 15: EtOH 2% PEG 3350 (0.6%) and PATPO3 PEG only +PEG1450b (0.6%)).

The various aspects of the invention described above can be combined toprovide further aspects of the invention. All of the references andproducts referred to in this application are in their entiretyincorporated herein by reference. Aspects of the invention can bemodified, if necessary to employ concepts of the references and/orproducts referred to in this application.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific aspects disclosed in thespecification and the claims, but should be construed to include allpossible aspects along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are limited by thedisclosure.

What is claimed is:
 1. A pharmaceutical composition consisting of PEG 400, PEG 3350, PEG 1450, butylated hydroxytoluene (BHT), methyl paraben, propyl paraben, and about 0.01% w/w to about 0.6% w/w isotretinoin, wherein the pharmaceutical composition has 0% w/w of ethanol and 0% w/w of water, and is formulated as a gel, ointment, lotion, emulsion, or cream.
 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has about 69% to about 70% w/w PEG 400, about 15% w/w PEG 3350 and about 15% w/w PEG
 1450. 3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition has about 0.2% w/w methyl paraben and about 0.02% w/w propyl paraben.
 4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition has about 0.1% w/w BHT.
 5. A pharmaceutical composition consisting of about 0.01% w/w to about 0.2% w/w isotretinoin, about 69% w/w to about 70% w/w PEG 400, about 15% w/w PEG 3350, about 15% w/w PEG 1450, about 0.1% w/w butylated hydroxytoluene (BHT), about 0.2% w/w methyl paraben, and about 0.02% w/w propyl paraben, wherein the pharmaceutical composition has 0% w/w of ethanol and 0% w/w of water, and is formulated as a gel, ointment, lotion, emulsion, or cream.
 6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition has about 0.01% w/w, about 0.025% w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, or about 0.2% w/w isotretinoin.
 7. A pharmaceutical composition consisting of 0.01% w/w to 0.2% w/w isotretinoin, 69% w/w to 70% w/w PEG 400, 14.78% w/w PEG 3350, 15% w/w PEG 1450, 0.1% w/w butylated hydroxytoluene (BHT), 0.2% w/w methyl paraben, and 0.02% w/w propyl paraben, wherein the pharmaceutical composition has 0% w/w of ethanol and 0% w/w of water, and is formulated as a gel, ointment, lotion, emulsion, or cream.
 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition has 0.01% w/w, 0.025% w/w, 0.05% w/w, 0.1% w/w, 0.15% w/w, or 0.2% w/w isotretinoin.
 9. A pharmaceutical composition consisting of PEG 400, PEG 3350, PEG 1450, butylated hydroxytoluene (BHT), methyl paraben, propyl paraben, and 0.01% w/w to 0.6% w/w isotretinoin, wherein the pharmaceutical composition has 0% w/w of ethanol and 0% w/w of water, and is formulated as a gel, ointment, lotion, emulsion, or cream.
 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition has 69% to 70% w/w PEG 400, 15% w/w PEG 3350 and 15% w/w PEG
 1450. 11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition has 0.2% w/w methyl paraben and 0.02% w/w propyl paraben.
 12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition has 0.1% w/w BHT. 